Production of 6-demethyltetracycline

ABSTRACT

This invention relates in an improved method for manufacturing antibiotics of the tetracycline group by growing a micro-organism of the class consisting of Streptomyces aureofaciens NC1B 9501, Streptromyces aureofaciens NC1B9502, and mutants and variants thereof aerobically in an aqueous fermentation medium containing a source of carbon and nitrogen and mineral salts, and recovering the antibiotic from the medium.

119 United States Patent [151 3,639,214 Mercer el al. 1 1 Feb. 1, 1972[54] PRODUCTION OF 6- 3,070,514 12/1962 Virgilio et a] ..l95/8ODEMETl-IYLTETRACYCLINE 3,092,556 6/1963 Growich et a1. 195/80 [72]Inventors: Clive Kenneth Mercer; George Desmond prim E l ryxammer-Joseph M. Gohan Wilkm, both of Nottingham, England AttorneyEugene 0 Renal. [73] Assignee: The Upjohn Company, Kalamazoo, Mich. [221Filed: June 4, 1964 [57] ABSTRACT [21] Appl' 372696 This inventionrelates in an improved method for manufacturing antibiotics of thetetracycline group by growing a micro- [52] 11.8. C1 ..195/80 Organismof the class consisting of Streptomyces aureofaciens [51] Int. Cl. r ,.C12d 9/18 NClB 9501, Streptromyces aureofaciens NC1B9502, and mu- [58]Field of Search 195/80 tants and variants thereof aerobically in anaqueous fermentation medium containing a source of carbon and nitrogenand [56] References Cited mineral salts, and recovering the antibioticfrom the medium.

UNITED STATES PATENTS 1 Claims, No Drawings 2,878,289 3/1959 McCormicket al. ..195/80 X PRODUCTION OF -DlEMETHYLTETRACYCLINE The compounds ofthe tetracycline group are very valuable broad-spectrum antibiotics, themost important members being tetracycline, chlortetracycline,oxytetracycline and 6- demethylchlortetracycline. Although tetracyclineis the parent compound of the group, chlortetracycline was the first oneto be isolated from fermentation brews of Streptomyces aureofaciens,followed by oxytetracycline. Tetracycline was first prepared on thelarge scale by catalytic hydrogenation of chlortetracycline althoughthere is usually a small proportion of tetracycline in chlortetracyclinebrews.

Following the initial isolation of tetracycline there have beendescribed numerous processes for the preparation of tetracycline byfermentation whereby a tetracycline and chlortetracycline producingorganism is grown in an aqueous nutrient medium which ischloride-reduced and the main product of the fermentation istetracycline. British Pat. Specification No. 775,115 claims a processfor the preparation of tetracycline by a chlortetracycline producingorganism in a medium in which the chloride ions have been reduced,possibly by ionexchange. British Pat. Specification No. 790,953 claims aprocess for preparing tetracycline by fermentation in a medium where thechloride ions have been reduced by dialysis. British Pat. SpecificationNo. 773,453 claims a process in which a chloride-free fermentationmedium is produced by precipitation of chloride ions with silvernitrate. British Pat. Specification No. 823,230 claims a process inwhich the tetracycline/chlortetracycline ratio in the fermentationproduct is increased by adding to a chloride containing medium from to500 parts per million of bromide ion. British Pat. Specification No.846,510 and British Pat. Specification No. 845,715 claim processes forthe fermentation of tetracycline in which the nutrient media containchlorination inhibitors.

These fermentation processes are costly in the preparation of chloridefree media or inefficient in that so much chlorination inhibitor isrequired that the tetracycline production by the organisms enzymes isappreciably reduced.

It is an object of the present invention to provide an improved processfor the fermentation of nonchlorinated tetracyclines in achloride-containing medium. It is a specific ob ject of the presentinvention to prepare tetracycline substantially free fromchlortetracycline by fermentation in a chloride-containing medium. It isa further specific object of the present invention to preparedemethyltetracycline substantially free from demethylchlortetracyclinein a chloridecontaining medium.

We have now discovered that by suitable mutation and selection ofStreptomyces aureofaciens we can produce a strain of the organismStreptomyces aureofacients TH.188, which when grown even in achloride-containing medium will produce tetracycline in high yield,substantially free of chlortetracycline. We have also discovered thatthis strain of Slreptomyces aureofaciens "fl-L188 and similarnonchlorinating organisms may be further mutated to give new strains,which when grown in a chloride containing medium will producesubstantially pure 6-demethyltetracycline. The preferred isolate isStreptomyces aureofaciens TK.232/ 2 for the preparation of-demethyltetracycline.

According to the present invention there is provided a process forpreparing tetracycline antibiotics of the general formula:

HO R H N(CH)2 CONHI l u OH O OH O wherein R is hydrogen or methyl, whichcomprises growing Streptomyces aureofaciens TH.188, TK.232/2 and mutantsthereof aerobically in an aqueous fermentation medium containing asource of carbon, nitrogen and mineral salts which need not bechloride-free or contain a chloride inhibitor.

The organism from which were derived the new mutants used in theprocesses according to the present invention was a normalchlortetracycline-producing strain of Slreptomyces aureofaciensdeposited at the culture collection of the North Regional ResearchLaboratories, Peoria, 111. United States of America, under the numberNRRL. B1286. The organism was mutated by irradiation with ultravioletlight until only about 0.1 percent of the organisms were viable.

After irradiation the remaining organisms were plated out and individualisolates were grown aerobically in shake flasks containing a normalchloride-containing medium suitable for the preparation ofchlortetracycline. The harvest brews were chromatographed and organismswere selected for their reduced ability to produce chlortetracycline.After a series of such mutations and selections we selected Strepmmycesaurofaciens Tl-l.188 which gave a high yield of tetracycline andsubstantially no chlortetracycline.

Using similar techniques of irradiation Streptomyces aureofaciens TH.47,a low-chlorinating organism obtained by irradiation of NRRL. B1286 inthe same way as the organism TH. 188, was mutated and the viable mutantswere selected for their ability to produce demethyltetracycline. Aftermutation and selection, an organism, Streptomyces aureafaciens TK.232/2was discovered which produced demethyltetracycline in fair yield,substantially free from tetracycline and chlorine-containingtetracyclines.

Streptomyces TH.l88 and Streptamyces TK.232/2 have been deposited at theNational Collection of Industrial Bacteria, Torry, Kincardineshire,Scotland, under the accession numbers NCIB 9501 and NCIB 9502respectively. The organisms will be referred to under their NCIBaccession numbers subsequently in this specification.

The mutated organisms have been selected by paper chromatography offermentation brews. Shake flask brews are harvested, the brew isextracted at PH2 with butanol and applied to a Whatman No. 1 paperpreviously dipped in a 0.1 molar aqueous solution of ethylenediaminetetraacetic acid and dried.

The paper is developed with the organic phase of a mixture of butanol (4parts), ammonium hydroxide 3.6. 0.88 (1 part), and water (5 parts) for18 hours and the position of the antibiotic is shown by irradiation ofthe dried paper with U.V. light.

For the separation of demethyltetracycline-containing brews, a similarchromatographic technique was employed but the solvent employed was amixture of chloroform (9 parts), butanol (1 part), saturated withaqueous 0.3 M. phosphoric acid containing 0.1 percent of trichloroaceticacid.

Typical Rfvalues in this system are:

chlortetracycline 0.78 b-Demethyltetracycline 0,71 Tetracycline 0,5 7 oDemethyltetracycline 0.40 Oxytetracycline 0.1 3

The mutants were selected using this system and the organ isms NCIB 9501and NCIB 9502 did not show the presence of chlorinated tetracyclines. Anaccurate assay of tetracyclines is not possible by this method andisolated solids from fermentations by the new mutants were assayed byquantitative biochromatogram.

Whole harvest brew is acidified with hydrochloric acid to pH 2 and isextracted with four-fifths volume of n-butanol. A single extraction ofthis nature gives an percent extraction of the active material in thebrew and the concentration in the butanol extract is thereforeequivalent "to the concentration in the initial brew. Whatman No. 1paper strips are soaked in 0.3 M Nal-l PO '2H O solution adjusted to pH3 with phosphoric acid and are then dried. The butanol extract (0.002ml.) is applied to the top of the strip and the chromatogram isdeveloped for 9 hours by the descending solvent phase using as solventbutanol saturated with 0.3 M NaH,PO,-2H,O solution at pH 3. Tetracyclineand chlortetracycline standard preparations are run concurrently. Astandard curve of chlortetracycline is prepared by chromatography ofpure, chlortetracycline free, tetracycline to which has been added knownamounts of pure chlortetracycline. The chromatograms are placed onbioplates seeded with Bacillus cereus and incubated overnight. The zonesof inhibition produced by chlortetracycline are measured and form thebasis for a standard curve such as is normally used in biological assay.Using this method roughly 0.1 percent of chlortetracycline has beendemonstrated in the brews according to the present invention.

The solids isolated from fermentation brews of mutant NCIB 9502 containsimilar negligible amounts of chlorinecontaining tetracyclines.Furthermore, neither of these organisms NCIB 9501 and NCIB 9502 producebromotetracyclines when grown in a medium containing bromide ions.

The mutant NCIB 9501 is characterized according to Bergey (7th Edition)as a strain of Streptomyces aureofaciens by the following criteria:

1. It is a mesophilic saprophyte.

2. It produces a soluble golden yellow pigment on some organic media.

3. Growth is orange in cinnamon with grey aerial mycelium.

4. It produces a yellowish pigment on synthetic media.

5. It produces a tetracycline.

The mutants NCIB 9501 and NCIB 9502 are grown initially on aconventional agar slant. A suitable composition is:

Asparagine 0.05% Beef extract 0.2% K,HPO 0.05% Glucose I .0% Agar l .7%Tapwater to 100% pH to 6.8 before sterilizing The slants prepared fromsuch a medium are inoculated with a spore suspension and incubated at 26C. for 14 days.

The growth from the agar slant is used to inoculate a standard primaryseed medium according to conventional antibiotic manufacturingtechnique. We find that a medium of the following constitution givesgood growth of the organism.

Corn steep liquor 3.2% vlv Sucrose 0.3% Chalk 0.5% Tapwater to 100% pH5.8-6.0 before sterilization Fifty milliliters of such a medium issterilized in a 250 ml. conical flask and inoculated with about 200 Xspores from an agar slant. Incubation is continued for 26 hours at 26 C.on a shaker with a speed of 250 r.p.m. and a throw of 2 inches. Thevegetative inoculum produced is then used to seed the main fermentationmedium at a concentration of 5 percent or a secondary seed using thesame primary seed medium.

The main fermentation stage is conveniently performed in a medium whichis suitable for the preparation of chlortetracycline and othertetracycline antibiotics but there is no necessity to reduce thechloride content of the medium nor to inhibit the use of chlorine by theorganism by addition of bromide ions, sulphonamides or by other chlorideion inhibitors. Suitable media include those with the followingcompositions:

Medium A Corn steep liquor 2.5-3.5% vlv Chalk 0.7-l.2% W/v Starch 3.0%Sucrose 3.5% Ammonium sulfate 0.6% Ammonium chloride 0.3% Manganoussulfate 0.0005-0.1% Lard Oil 1.6% Tapwater to 100% pH before sterilizing5.8-6.2 Medium B Similar to medium A but also including Pharmamedia 0.6percent a commercially available protein hydrolysate. Medium C Glucose2% Soya flour 1% Corn steep liquor 1% Sodium chloride 0.5% Calciumcarbonate 0.5% Tapwater to pH before sterilizing 6.6-6.8

We have found that Media A and B give particularly good yields oftetracycline and that it is very important that manganese should bepresent.

Absence of manganese from the medium reduces the tetracycline titre ofan otherwise typical fermentation to below 30 percent of the normalvalue. The preferred Medium D is one of type B containing 2.5 percentv/v corn steep liquor, 1.0 percent chalk and 0.01 percent manganoussulphate.

Fermentation conditions are similar to those customarily used in thegrowth of Streptomyces aureofaciens. The aerobic fermentation ismaintained at about 26 C. for 6-7 days with additions of an antifoamingagent if required. Suitable antifoarn agents are cetyl alcohol andtriethanolamine stearate which may conveniently be added in solution inlard oil.

When the fermentation has reached its peak yield of antibiotic, the brewis harvested and the antibiotic isolated by known methods.

The invention is illustrated by the following nonlimitative examples.

EXAMPLE l An 8 l. stirred ferrnenter containing sterile Medium D (4 l.)was inoculated with a 200 ml. portion of a secondary seed vegetativeinoculum of Streptomyces aureofaciens NCIB 9501. Fermentation at 26 C.was continued with a stirrer speed of 700 r.p.m. for 6 days. Duringfermentation sterile air was introduced at a rate of 8 liters/minute.The brew was adjusted to pH to 2.5 with hydrochloric acid, diluted with2 volumes of water, the mycelium was filtered off, washed with acidwater, filtered, and the filtrates were combined. There was isolatedfrom the combined filtrates 3.95 g. of tetracycline, assaying 1020 g/gby bioassay and containing only 0.13 percent chlortetracycline(quantitative biochromatogram).

EXAMPLE 2 A shake flask containing 40 ml. of Medium D was inoculatedwith 1.3 ml. of a primary seed vegetative of Streptomyces aureafaciensNCIB 9502, and was fermented for 6 days at 26 C. at a shaker speed of250 r.p.m. with a throw of 2 inches. The fermentation brew from a numberof such shake flasks was combined and a bulked acid filtrate obtained asin example 1.

From the bulked filtrate there was isolated 6- demethylchlortetracyclinewhich was free of tetracycline and -demethylchlortetracycline whentested by paper chromatography.

EXAMPLE 3 An 8 litre stirred fennenter containing sterile medium D (41.) was inoculated with ml. of a 24-hour secondary seed vegetativeinoculum of Streptamyces aureofaciens NCIB 9502 and was fermented at 26C. for 6 days with a stirrer speed of 700 r.p.m. Sterile air wasintroduced at a rate of 4.5 l./min. Whole brew from these fermentations(10 1.) was acidified to pH 1.5 with concentrated hydrochloric acid,kieselguhr was added, the mixture was filtered and the mycelium waswashed with water (2X2 1.). The combined filtrates were adjusted to pH8.8 with sodium hydroxide, 80 percent Sequestrol Na (15 ml.) was addedand the filtrate was extracted with butanol (2 1.). The aqueous liquorwas reextracted twice with butanol (1 the extracts were combined andwere extracted with dilute sulphuric acid at pH 1.5 (4X300 ml.). Thecombined acid extracts were concentrated in vacuo to 200 ml., sodiumhydroxide was added to pH 5 and the solution was left to standovernight. The crystals of crude antibiotic which separated werecollected by filtration, washed with water and dried in vacuo.

The crude base was dissolved in cold methanol at the rate of 40 ml./g.,was filtered through Kieselguhr and concentrated to small volume.Addition of a few drops of water produced flesh colored crystals whichwere collected by filtration and dried to give -demethyltetracyclinedihydrate m.p. l75-180 C. (d), [ad -270 (1 percent in N/lO HCI). (Found:C, 53.8; H, 5.7% C H NO -ZH O requires C, 54.1 H, 5.6 percent).

The recrystallized base 2.5 g.) was dissolved in cold methanol (100 ml.)and neutralized with concentrated hydrochloric acid (0.65 ml.) Thesolution was filtered through Norit SX Plus charcoal (l g.) which hadbeen previously washed with methanolic HCl and the pale yellow filtratewas concentrated in vacuo to a small volume. Replacement distillationwith acetone gave dull yellow crystals of o-demethyltetracyclinehydrochlorine sesquihydrate, decomposed at about 210 C. [ab-259 (1percent N/lO HCl). (Found: C, 51.2; H, 5.3% C ll ClN O 1% H O requiresC, 51.1; 5.3 percent).

The crude base (0.5 g.) was suspended in ethanol ml.) andtoluene'p-sulphonic acid monohydrate (0.4 g.) was added. A clearsolution was obtained from which beige colored plate crystals rapidlyseparated. These were collected by filtration, washed with ethanol andrecrystallized by dissolving in methanol (20 ml.) containingtoluene-p-sulphonic acid (0.] g.), filtering through charcoal which hadbeen acid washed and diluting the filtrate with ether. The pale yellowneedles which crystallized were collected to @ve 6-demethyltetracyclinetoluene-p-sulphonate m.p. 230C. (d) [a],,-2l3 (1% W10 HlCl). (Found: C,55.9; H, 5.1% c,,n,,,r-1,o,,s requires C, 55.8 H, 5.0 percent).

When subjected to thin layer chromatography on a silica gel coveredplate which had been prerun with methanol containing 10 percent v/vconcentrated hydrochloric acid to remove iron, the chromatogram beingdeveloped by the same solvent, the free base, hydrochloride andtoluene-p-sulphonate all give a single spot.

We claim:

l. The process of producing Gdemethyltetracycline which comprisesgrowing a micro-organism selected from the class consisting ofStreptomyces aureofaciens NClB 9502 and nonchlorinating-demethyltetracycline producing mutants and variants thereof undersubmerged aerobic conditions in an aqueous fermentation mediumcontaining a source of carbon and nitrogen and mineral salts, andrecovering 6- demethyltetracycline from the medium.

